Preparation of samples for proteome analysis

Inactive Publication Date: 2010-12-09
PRONOTA NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0010]Given that each protein present in a sample includes an N-terminus and a C-terminus, enrichment for N-terminal peptides or for C-terminal peptides achieves excellent representation of the majority of proteins of said sample and, at the same time, considerable reduction of the complexity of peptide mixtures to be analysed. Also, the present methods can be applied on whole peptide digests, which considerably simplifies the preparation of samples for protein profiling and proteome analysis.
[0042]Otherwise, it is also contemplated to first separate (fractionate) the protein peptide mixture into fractions of peptides using the above described separation step, and only thereafter treat said fraction(s) with aminopeptidase or carboxypeptidase to isolate N-terminal peptides or C-terminal peptides there from, respectively. Such sequence of actions may, e.g., allow to perform the reaction with amino- or carboxypeptidase on a limited number of fractions of interest, thereby reducing the reaction volumes and need for reagents.

Problems solved by technology

However, proteolysis of complex biological samples can produce thousands of peptides, which may overwhelm the resolution capacity of current chromatographic and mass spectrometric systems, causing incomplete coverage and impaired identification of the constituent peptides.
Although valuable, the method involves multiple handling steps, which might introduce errors and increase labour-intensiveness.

Method used

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  • Preparation of samples for proteome analysis
  • Preparation of samples for proteome analysis
  • Preparation of samples for proteome analysis

Examples

Experimental program
Comparison scheme
Effect test

example 1

Acetylation Protects Peptides from Degradation by Aminopeptidase

[0135]In the first experiment we investigated the effect of using acetylation as N-terminal blocking on progressive hydrolysis of peptides by bacterial aminopeptidase. As substrate for aminopeptidase activity we used the unprotected peptides from the Pepmix4 calibration mix (PepMix4, LaserBioLabs #C104). One tube was dissolved in 100 μl water resulting in peptide concentrations ranging form 8-50 pmoles / μl. As protected peptides we used 2 acetylated peptides designated P1380 and P1384 (for sequences see table 1). Both were diluted to 40 pmoles / μl. The sample reaction mixture that was used contained 1 μl of PepMix4 and 1 μl of each P1380 and P1384, all added to 20 mM TrisHCl buffer pH8, to a final volume of 20 μl. One sample was incubated overnight at 37° C. with 1 unit of Aeromonas proteolytica aminopeptidase (Sigma-aldrich, A8200) while another sample was left untreated. The reaction was stopped with 10% Trifluoroacetic...

example 2

ICPL Leads to N-terminal Protection from Aminopeptidase Activity

[0136]We investigated whether other blocking groups could improve the protection from aminopeptidase activity. In this experiment we also studied the use of Aminopeptidase M purified form pig kidney microsomes.

[0137]To prevent contamination issues with the commercial aminopeptidase M preparation (Sigma Aldrich, #L5006), the enzyme batch was dialyzed against 60 mM Sodiumphosphate at pH7. The ICPL reagent (Serva ICPL™-kit, #39230) was employed as a blocking agent to improve the protection from aminopeptidase activity. The rationale behind this is the greater sterical hindrance for enzyme activity if a bigger group is introduced on the N-terminus. ICPL labeling of 4 peptides designated L2 to L5 (Table 1), was performed according to the manufacturer's instructions. A mixture was prepared with 200 pmoles of the Pepmix4 peptides, 50 pmoles of each L2 to L5 and 0.05U of the dialyzed aminopeptidase M. An overnight incubation at...

example 3

The Modified N-terminal Peptides of Proteins are more Resistant to Aminopeptidase Activity when Compared to their Internal Peptides

[0138]This experiment was performed to show that the N-terminal peptides can be enriched by their altered susceptibility to aminopeptidase activity.

[0139]For this analysis, we prepared a mixture of 7 proteins (α-1-antitrypsin, hemoglobin, transferrin, albumin, β-lactoglobulin, α-1-acid glycoprotein and catalase; all from Sigma-aldrich) in PBS containing 4M Guanidine HCl. A total of 0.7 mg protein (0.1 mg / protein) was used for sample preparation. The sample was first reduced by treatment for 10′ at 30° C. with 6 μl 0.1M Tris(2-carboxyethyl)-phosphine Hydrochloride (TCEP.HCl, Pierce, #20490) after adjusting to pH 7.0. After this reduction, the free sulfhydryl groups on cysteines are alkylated by adding 6 μl 0.2M iodoacetamide (Fluka, #57670) for 60′ at 30° C. The sample was subsequently brought on a NAP™5 gelfiltration column (GE healthcare, #17-0853-02) t...

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Abstract

The invention mainly concerns methods and systems for the preparation of biological samples for proteome analysis, N-terminal or C-terminal peptides of proteins are enriched using exopeptidase.

Description

FIELD OF THE INVENTION[0001]The invention relates to the field of proteomics. Methods and systems for the preparation of biological samples for proteome analysis, and for identification and quantification of proteins and peptides from such samples are provided.BACKGROUND OF THE INVENTION[0002]The proteome is usually described as the entire complement of proteins found in a biological system, such as, e.g., a cell, tissue, organ or organism. Proteomics is concerned with the study of the proteome expressed at particular times and / or under internal or external conditions of interest. Proteomics approaches frequently aim at global analysis of the proteome, and require that large numbers of proteins, e.g., hundreds or thousands, can be routinely resolved and identified from a single sample.[0003]Among the promises of proteomics is its ability to recognise new biomarkers, i.e., biological indicators that signal a changed physiological state, such as due to a disease or a therapeutic inter...

Claims

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Application Information

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IPC IPC(8): C12P21/00C12N9/48
CPCG01N33/6842C07K1/12
Inventor EYCKERMAN, SVENKAS, KOEN
Owner PRONOTA NV
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